Figure 5.

Lysine 357 can be acetylated in vivo. (A) H1299 cells were transfected with WT-p53 and either p300-HA, CBP-HA, or pCAF-Flag expression plasmids. Cells were lysed and proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Immunoblot analysis with indicated antibodies was performed. (B) H1299 cells were transfected with CBP-HA and either WT-p53 or 2KR-p53. After 24 hours, cells were lysed and analyzed as in A. (C) HCT116 cells expressing p53 (+/+) were treated with 5FU (0.5 mM ) for 24 hours or MG132 (25 uM) for 6 hours, lysed, and subjected to immunoblot analysis with antibodies recognizing p53, p53-Ac357, or actin. HCT116 p53-null cells (−/−) were run alongside as a control. (D) H1299 cells were transfected with constructs expressing single tetramerization domain lysine p53 mutants as indicated. Cells were lysed and immunoblot analysis to detect p53 level and actin as indicated. (E) Parallel cultures of H1299 cells were treated as in D. RNA was extracted and analyzed by qPCR for p21 expression, which was normalized to hprt1 levels. Error bars are the standard deviation of 3 experiments.